IL-12和IL-15对外周血CIK细胞增殖和对SMMC-7721肝癌细胞杀瘤效果研究

Study on the effect of IL-12 and IL-15 on the proliferation of CIK cells from of peripheral blood and the activity of killing SMMC-7721 hepatoma cell line in vitro

目的观察常规培养条件下添加白细胞介素(IL)-12、IL-15对细胞因子诱导的杀伤(CIK)细胞增殖的影响,观察不同细胞因子组合培养的CIK细胞的杀瘤作用,为高效率、高质量体外制备肿瘤患者自体CIK细胞制剂提供新思路。方法首先筛选出IL-2、IL-12、IL-15最佳添加水平;然后采集健康献血员外周血,分离出单个核细胞后,根据添加的细胞因子种类和组合类型进行分组:A组(IL-2常规培养组)、B组(IL-2^+IL-12组)、C组(IL-2^+IL-15组)、D组(IL-2^+IL-12^+IL-15组)、E组(细胞因子空白对照组);各组单个核细胞分别在培养第0、5、10、15、20天,采用全自动血球计数仪进行CIK细胞计数,锥虫蓝染法检测细胞活性,流式细胞技术检测细胞膜CD3、CD8、CD56阳性率,MTS法测定CIK细胞对肝癌SMMC-7721细胞的杀瘤效果。结果B、C、D组培养第10、15、20天后CIK细胞增殖倍数均明显高于A组(P<0.05);D组培养第10、15、20天CIK细胞增殖倍数均分别明显高于C组(P<0.05)。B、C、D组培养第15、20天CIK细胞膜CD3^+CD8^+、CD3^+CD56^+百分比均分别明显高于A组(P<0.05);D组培养第15、20天CIK细胞膜CD3^+CD8^+、CD3^+CD56^+百分比均分别明显高于B组(P<0.05)。在效靶比5∶1时,各组培养第10、15、20天的CIK细胞对肝癌SMMC-7721细胞的杀伤率均明显高于A组(P<0.05);D、C组培养第10、15、20天CIK细胞对肝癌SMMC-7721细胞的杀伤率分别明显高于B组(P<0.05)。结论 IL-12、IL-15均可促进CIK细胞增殖,IL-15在促进CIK细胞增殖的同时还能增强CIK细胞杀伤肝癌SMMC-7721细胞的活性。

Objective To observe the influence of IL-12,IL-15 on CIK cell in the normal culture;to observe the anti-tumor effect in the circumstance of different combination of cytokines,and to provide a new insight for preparing high effective and qualified CIK cell in vitro.Methods The optimal concentrations of IL-2,IL-12 and IL-15 were determined,respectively.After the peripheral blood from healthy blood donors was collected,monocytes were selected and co-cultured with different cytokines into different groups,as group A（IL-2 normal culture group）,group B（IL-2 and IL-12 group）,group C（IL-2 and IL-15 group）,group D（IL-2,IL-12 and IL-15 group）,and group E（cytokine control group）.The monocytes in different groups were calculated by globulimeter,the activity of cells was detected by Trypan blue staining,positive ratio of CD3,CD8,CD56 on the celluar membrane was detected by flow cytometry,and the anti-tumor effect of CIK to SMMC-7721 was detected by MTS method,in the day of 0,5,10,15,20 after the culture.Results Statistical analysis indicated that,the proliferation multiplication of CIK cells was significantly higher in group B,group C and group D after 10,15 and 20 days of culture than those in group A（P〈0.05）;and group D had higher proliferation multiplication than that of group C（P〈0.05）.The percentage of CD3~＋CD8~＋,CD3~＋CD56~＋in CIK cell membrane in group B,C,D was significantly higher than that in group A after 15 and 20 days of culture（P〈0.05）.The percentage of CD3~＋CD8~＋,CD3~＋CD56~＋in CIK cell membrane in group D was significantly higher than that in group B after 15 and 20 days of culture（P〈0.05）.The killing rate of CIK cells for liver cancer in each group at 10,15,20 days of culture was significantly higher than that of group A when the target target ratio was 5∶1（P〈0.05）.The killing rate of CIK cells for liver cancer in group D,C at 10,15,20 days of culture was significantly higher than that of group B（P〈0.05）.Conclusion IL-12 and IL-15 could improve the proliferation of CIK cells,and IL-15 also has the effect of enhancing CIK cells the tumor-killing to SMMC-7721 activity.